More than a decade ago, we reported that among dendritic cells, EpCAM was selectively expressed by epidermal Langerhans cells. In the past few years, we have revisited the utility of EpCAM as Langerhans cell surface marker and have extended our earlier studies and demonstrated that EpCAM expression, in conjunction with other surface markers, differentiates Langerhance cells from all other dendritic cells, including several recently described novel cutaneous dendritic cell subsets. In an effort to elucidate important aspects of EpCAM function in vivo, we identified a pre-existing targeted mouse embryonic stem cell generated by BayGenomics and, working with the CCR Mouse Knockout Core Facility headed by Dr. Lino Tessarollo, generated EpCAM +/- mice in which beta-galactosidase was inserted into one EpCAM allelle. EpCAM +/- mice were viable, fertile and indistinguishable from wild type littermates. Examination of beta-galactosidase expression as a surrogate for EpCAM revealed that EpCAM was expressed in a variety of developing epitehelial structures in skin and other organs. Mating of EpCAM +/- male and female mice gave rise to only wild type and heterozygous animals. No viable EpCAM-deficient pups were obtained. Assessment of embryos in timed pregnant females revealed that EpCAM-deficient embryos implanted and were indistinguishable from EpCAM-sufficient embryos until EGA 8.5 when they began to exhibit developmental delay. EpCAM-deficient embryos became nonviable and were resorbed soon thereafter. We subsequently determined that EpCAM was transiently expressed in conceptus-derived placentas with maximal expression at EGA 8.5-9.5. Detailed studies of placentas associated with EpCAM-deficient embryos revealed that they were small and thin with incompletely developed and poorly vascularized labyrinthine layers. EpCAM-deficient placentas also contained markedly decreased numbers of parietal trophoblast giant cells, a phenotype that has previously been associated with embryonic lethality. The findings were reported in PLoS One in 2009. Thus, although mechanistic aspects of EpCAM function remain to be elucidated, EpCAM clearly has one or more nonredundant roles in normal physiology. To gain additional insights into EpCAM function, we have generated mice with an EpCAM allele that can be conditionally deleted in a lineage-specific fashion. Working with Dr. Tessarollo and using recombineering, we developed a targeting vector that allowed loxp sites to be inserted into the EpCAM locus. Embryonic stem cells with a targeted EpCAM allelle were generated and identified, and then utilized to generate the corresponding mice. Germline transmission of the targeted allele has been confirmed. We have now crossed mice with targeted EpCAM alleles with mice that express the recombinase cre in cells of various lineages to ask and answer relevant questions. Tissues or cells of interest include Langerhans cells, keratinocytes, thymic epithelial cells and intestinal epithelia. The latter tissue is of interest because EpCAM mutations have been causally linked to congential tufting enteropathy, a rare congenital diarrheal syndrome. Experiments that have been completed to date clearly indicate that the targeted EpCAM allele efficiently recombines in several lineages and that this results in phenotypes in several tissues. We are characterizing these phenotypes and are carrying out complementary in vitro studies to gain additional insights into mechanistic aspects of EpCAM function. An initial series of investigations regarding EpCAM function in Langerhans cells has been completed and the findings were published in the Proceedings of the National Academy of Science in 2012. We determined that Langerhans cells that fail to express EpCAM exhibit decreased motility in epidermis and exit epidermis more slowly than EpCAM-expressing Langerhans cells after antigen-induced activation. This results in decreased migration of Langerhans cells from skin to regional lymph nodes and enhanced contact sensitivity reactions. These results definitively linking Langerhans cell migration to function and support the concept that Langerhans cells have a anti-inflammatory function as suggested by others. The results also suggest that EpCAM may be anti-adhesive, rather than pro-adhesive as previously suggested. In studies that are ongoing, we are additionally characterizing the role of EpCAM in Langerhans cell-keratinocyte interactions and in additional immune-related functions. Subsequent studies in mice whose Langerhans cells lacked EpCAM revealed that antibody-forming responses to proteins that were applied to skin were enhanced rather than predicted. Increased immune responses were associated with enhanced localization of antigen-bearing Langerhans cells in skin draining lymph nodes. These results have recently been published in the Journal of Investigative Dermatology. Our interpretation of the our seeming disparate results using mice whose Langerhans cells lack EpCAM is that the role that EpCAM plays in the regulation of Langerhans cells is context dependent. We have also explored mechanisms by which EpCAM regulates intercellular adhesion using human intestinal epithelial cells as a model system. We have determined that EpCAM acts in part by binding tightly to the tight junction-associated protein claudin-7. This interaction sequesters claudin-7 distinctly away from tight junctions and protects claudin-7 and associated claudin-1 from degradation in lysosomes. In the absence of EpCAM, whole cell levels of claudin-7 and claudin-1 decrease dramatically but remaining claudin-7 and claudin-1 becomes tight junction-associated. This results in increased tight junction avidity and increased trans-epithelial electrical resistance. These findings have been described in a manuscript that has been submitted for publication. These results have recently been published in the Journal of Biological Chemistry. Studies intended to delineate the mechanism(s) by which EpCAM stabilizes selected claudins are in progress. We have also begun to propagate mouse intestinal organoids and spheroids and are we assessing EpCAM-function in the these model system. We have observed a dramatic phenotype that can be reversed to large extent by treatment with a single pharmacologic inhibitor. The mechanism(s) that are responsible for this effect are under investigation, but our studies clearly argue for an important role in epithelial stem cell survival an/or proliferation. We have also identified and characterized a novel proteolytic pathway that is a dominant regulator of EpCAM function. This novelpathway has been described in a publication that has appeared in the Journal of Clinical Investigation.. In an effort to explore EpCAM function in other tissues, we are involved in collaborations with several developmental biologists with relevant expertise. One question that we are addressing relates to the functional equivalence of EpCAM and TROP2, an EpCAM-related molecule that is also expressed in epithelial tissues. The approach that we are taking involves use of transgenic mice that express EpCAM or TROP2 in intestinal epithelium and mice that have germline null mutations in EpCAM.